Transposable elements are DNA sequences that can move (i.e., transpose) to different genomic locations and they are present in the genomes of virtually all organisms studied. There are two general classes of transposable elements: transposons and retrotransposons. Retrotransposons mobilize via an RNA intermediate and transpose by a replicative mechanism termed retrotransposition. Long Interspersed Nuclear Elements (LINES or L1s) are the most abundant retrotransposons in the human genome and they comprise approximately 15 percent of DNA. An estimated 30-60 human L1 elements are retrotransposition-competent, and both germ line and somatic L1 retrotransposition events have caused disease. It also is hypothesized that the proteins encoded by retrotransposition-competent L1s mobilize certain SINES (e.g., Alu elements) and processed pseudogenes, which comprise another 10 percent of human DNA. Alu retrotransposition also is mutagenic and eleven de novo Alu insertions have been found to cause different diseases in the past seven years. Thus, either directly or through the promiscuous mobilization of cellular RNAs, L1s are potent mutagens in the human genome. Despite the mutagenic potential of L1s, studies concerning the mechanistic aspects of L1 retrotransposition are in their infancy. A recently developed assay to monitor L1 retrotransposition in cultured human cells and molecular biological and biochemical approaches will be used to determine the mechanism of L1 retrotransposition at the molecular level. Specifically, experiments will be performed to: 1) identify cis-acting sequences in L1 RNA and function al domains in the L1-encoded proteins required for retrotransposition; 2) identify factors that govern template choice by the L1-encoded proteins; and 3) identify in vivo intermediates in the L1 retrotransposition pathway. The long-term goal of this project is to gain a fundamental understanding of how L1 retrotransposition contributes to human disease and genetic diversity. A fundamental mechanistic understanding of L1 retrotransposition also will allow the practical development of engineered L1s as a transposon mutagen for the mouse genome and as a potential gene delivery vehicle for human studies.